The four species of chlamydiae cause a variety of serious diseases in both humans and in many animal species. The most serious chlamydial diseases of humans include trachoma, pneumonia, and a spectrum of different sexually transmitted conditions. Millions of people are debilitated by these diseases worldwide. The chlamydiae are obligate intracellular bacterial pathogens that develop within a non-acidified vacuole (the inclusion) within infected host cells. The biology of inclusion formation and development is only beginning to be understood. A collection of proteins produced by Chlamydia psittaci (IncA, IncB, IncC) are localized to the inclusion membrane during chlamydial development. These are the only known proteins, of either chlamydial or host origin, that are localized to the membrane of the chlamydial inclusion. Recently IncA was shown to be phosphorylated by host cell enzymes and exposed to the cytoplasm at the surface of the inclusion. These results suggest IncA may be important in the interaction of the inclusion with its intracellular environment. Functional characterization of IncA/B/C is complicated by the lack of a genetic system in the chlamydiae, as well as the absence of similarity between these proteins and any sequences in the databases. The overall goal of this research proposal is to identify the functions of IncA/B/C in the chlamydial developmental process. The three Aims in this proposal describe methodologies designed to achieve that goal. The experiments proposed in Aim 1 are designed to identify domains of IncA/B/C exposed to the cytoplasm in infected cells. Aim 2 utilizes the yeast two-hybrid system and bacteriophage display methodologies to examine interactions between IncA/B/C and proteins of either host or chlamydial origin that may be present in the cytoplasm or within the inclusion. Aim 3 describes the use of two expression systems, vaccinia virus and eukaryotic expression plasmids, to examine and mutagenize incA/B/C within host cells. These expression experiments will be conducted both in the absence or the presence of a parallel chlamydial infection. These different approaches will examine the unique biology of IncA/B/C as well as explore new technologies for investigating chlamydial interactions with mammalian cells.